Oxidants accumulate at sites of tissue inflammation involving neutrophils. Cochrane et al., J. Clin. Invest. 71:754-61 (1983); Ward et al., J. Clin. Invest. 72:789-801 (1983). The ability of antioxidants to block physiologic and morphologic disruption at these sites suggests that oxidants contribute to neutrophil-associated tissue injury. Ward et al., J. Clin. Invest. 72:789-801 (1983); Till et al., J. Clin. Invest. 69:1126-35 (1982). However, oxidants probably cooperate with other inflammatory effector agents, including proteases, to cause tissue injury in vivo. Schraufstatter et al., J. Clin. Invest. 73:1175-84 (1984); Revak et al. J. Clin. Invest. 76(3):1182-92; Lee et al. N. Engl. J. Med. 304:192-196 (1981); McGuire et al. J. Clin. Invest. 69:543-53 (1982). In order to assay the effects of antioxidants and antiproteases in experimental animals and humans, effector-specific biochemical markers of tissue injury are needed. Ideally, those markers should be soluble and present in blood and/or other accessible body fluids.
Fibronectins are large glycoproteins found in plasma, on cell surfaces, and in extracellular matrices. By binding other macromolecules as well as cells, they serve to promote anchorage of cells to substrata. Cochrane et al., J. Clin. Invest. 1:754-61 (1983); Hynes et al. J. Cell Biol. 5:369-77 (1982). Fibronectins are composed of subunits of variable primary structure [average relative molecular mass 250 kilodaltons (kDa)], that are disulfide-linked to form dimers or multimers. Hynes, in Cell Biology of the Extracellular Matrix, Hay ed., Plenum Press, pages 295-334 (1982); Hynes, et al., Cell Biol. 95:369-77 (1982); Ruoslahti et al. Meth. Enzy. 82:803-30 (1982); Schwarzbauer et al. Proc. Natl. Acad. Sci. USA 82:1424-28; Kornblihtt et al. EMBO J., 4(7):1755-59 (1985). Plasma fibronectin (PFn) is secreted by hepatocytes, whereas cellular fibronectin (CFn) is secreted by a variety of cultured cells including endothelial cells and fibroblasts. Tamkun et al. J. Biol. Chem. 58 (7):4641-47; Jaffe et al. J. Exp. Med. 147:1779-91 (1978); Birdwell, et al. Biochem. Biophys. Res. Commun. 97(2):574-8 (1980). Despite extensive physical and immunologic similarities, the two classes of fibronectin differ in electrophoretic behavior, solubility, and biologic activities. Tamkun et al., J. Biol. Chem. 258(7):4641-47 (1983); Yamada et al., J. Cell Biol. 80:492-98 (1979); Yamada et al. Biochemistry 16(25):2552-59 (1977).
Primary structural differences between plasma and cellular fibronectins have been found by peptide mapping [Hayashi et al. J. Biol. Chem. 256(21):11,292-11,300 (1981)] and immunologic techniques [Atherton et al. Cell 25:133-41 (1981)]. Recently, a difference region encoding for exactly one 90 amino acid type III structural repeat was identified in mRNA from human fibroblasts and two human tumor cell lines, but could not be detected in human liver mRNA. Kornblihtt et al. EMBO J. 4(7):1755-59 (1985); Kornblihtt et al., EMBO J. 3(1):221-26 (1984); Kornblihtt et al., Nucleic Acids Res. 12(14):5853-68 (1984). Since plasma fibronectin is synthesized by hepatocytes, it is likely that the extra type III repeat is a unique domain of cellular fibronectins. Schwarzbauer et al., Proc. Natl. Acad. Sci. USA., 82:1424-28 (1985); Kornblihtt et al., EMBO J. 3(1):221-26 (1984); Kornblihtt et al., Nucleic Acids Res. 12(14):5853-68 (1984).
Fibronectin accumulates at sites of injury and inflammation in vivo [Pettersson et al., Clin. Immunol. Immunopath 11:425-436 (1978); Grinnel et al. J. Invest. Derm. 76:181-189 (1981); Repesh et al. J. Histochem. Cytochem. 30(4):351-58 (1982); Torikata et al., Lab. Invest. 52(4):399-408 (1985); Carsons et al. Arth. Rheum 24(10):1261-67 (1981)] and is produced by cells in blood vessel walls at these sites. Clark et al., J. Exp. Med. 156:646-51 (1982); Clark et al., J. Immunol. 126(2):787-93 (1981); Clark et al., J. Invest. Derm. 79:269-76 (1982); Clark et al., J. Clin. Invest. 74:1011-16 (1984).
Cellular fibronectin might accumulate in the vascular compartment and in other fluids in communication with sites of inflammatory tissue injury. Studies discussed in detail hereinafter were carried out using mediators of inflammatory tissue injury, including oxidants and proteases, to promote in situ release of cellular fibronectin. We chose to perform our studies in an in vitro perfused rabbit lung, since this model provides an abundantly vascular tissue that can be observed for both physiologic and biochemical changes under defined conditions.